Diffuse intrinsic pontine glioma (DIPG) is an aggressive and incurable pediatric brain tumor that remains understudied. No progress in treatment has been made in the past 30 years, and radiation remains the standard of care. Recent whole-genome sequencing on tumor samples revealed heterozygous lysine-to-methionine mutations of histone variant H3.3 at amino acid residue 27(K27M) in 80% of patients. We and others have shown that K27M invokes a gain-of-function mutation which inhibits the histone methyltransferase EZH2. This inhibition causes an overall global loss of the H3K27me3 mark as well as a restructuring of the residual H3K27me3 landscape. Chromatin immunopreciptiation-sequencing (ChIP-seq) analysis of human tumors with or without K27M mutations revealed the Cdkn2a transcript variant p16 as a target of increased H3K27me3. Expression analysis of our murine H3.3K27M glioma model recapitulated these results and additionally revealed hoxa4, 5, and 7 among the most significantly downregulated genes. Since the tumor suppressor p16 is mutated in several cancers but rarely appears in DIPG, we hypothesize K27M mutations drive tumorgenesis in part by epigenetically silencing this locus. Alteration of the hox locus has been implicated in DIPG due to its role during brainstem development. Our results suggest K27M mutation inhibits differentiation of brainstem progenitors by repressing these genes. To investigate these hypotheses I propose to characterize the H3K27me3 landscape at the Cdkn2a and Hoxa loci in our murine H3.3K27M glioma model. Additionally, I will take advantage of our conditional Cdkn2a floxed model to determine the relationship between H3.3K27M mutant expression and the p16 tumor suppressor pathway with regard to oncogenic transformation. The goal of this project is to determine how mutant H3.3K27M drives tumorigenesis by manipulating the epigenome to repress the p16 and Hoxa genes. The repression of H3K27me3 at these sites may be enhancing cell cycle progression and maintaining a primitive progenitor-like state and targeting these genes with chemotherapeutics could lead to a more efficacious treatment.